1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns nucleic acid segments isolated from human and murine sources, which encode a male germ cell specific protein, designated MORC. Various methods for making and using MORC DNA segments, DNA segments encoding synthetically-modified MORC proteins, and native and synthetic MORC polypeptides are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various immunological and diagnostic applications. Also disclosed are methods for identifying MORC-related polynucleotides and polypeptides, and methods for diagnosing and treating infertility or cancer, and in particular, testicular cancer, as well as screening methods for compounds that are involved with the development of cancer or spermatogenesis.
1.2 Description of Related Art
The genetic control of spermatogenesis is complex (Sassone-Corsi, 1997). Mutations at multiple loci and in structurally and functionally disparate genes in the mouse genome affect gametogenesis (Handel, 1987). Most mutations are pleiotropic, causing multi-system pathologies rather than isolated spermatogenic abnormalities. For example, the autosomal recessive mutation weaver, which results in degeneration of germ cells, also causes loss of the cerebellar granular cell layer and ataxia in affected mice (Vogelweid et al., 1993). The histologic phenotypes of mutations that affect germ cells are varied and include both reduced cell numbers and abnormal cell morphologies. Further complicating the understanding of germ cell biology is the fact that genes known to be essential for spermatogenesis participate in multiple cellular processes, including transcriptional control (Nantel et al., 1996; Blendy et al., 1996), cell proliferation (Toscani et al., 1997), protein folding (Dix et al., 1996) and DNA repair (Baker et al., 1995; Donehower et al., 1992).
1.2.1 Genetic Control of Mouse Spermatogenesis
The genetic control of mouse spermatogenesis has been extensively reviewed in the literature (Handel, 1987). Briefly, spermatogenesis is a complex and highly ordered developmental process, lasting 36 days in mice. Three phases of spermatogenesis can be distinguished: mitotic proliferation and renewal of spermatogonia, or stem cells; meiotic reduction division of spermatocytes; and differentiation of haploid spermatids into mature sperm cells, or spermiogenesis. The first meiotic division in protracted, with cells remaining in pachytene stage for 11 days. During this time, homologous chromosomes pair and recombine, and there is extensive DNA repair synthesis and transcription. Many genes must act during this stage of spermatogenesis, and it is the target of a number of mutations.
1.2.2 Mouse Spermatogenesis Mutations
A large number of spontaneous and induced mouse mutations resulting in abnormalities of normal spermatogenesis and fertility have been identified (Table 1). Recent reviews cataloging these mutations demonstrate the genetic heterogeneity and phenotypic pleiotropy of infertility (Handel, 1987; Chugg , 1989; Simoni, 1994; Wilmut et al., 1991). Mutations can be divided into three general groups. Pre-testicular phenotypes are the result of pituitary abnormalities or improper embryogenesis and germ cell migration (e.g., gcd). Intratesticular phenotypes result from mutations which manifest as abnormalities of the germ cells themselves (spermatogonia, spermatocytes, spermatids) (e.g., dazla). Post-testicular phenotypes encompass mutations that produce spermatozoa with abnormal function (e.g., hotfoot).
The majority of the mutations in Table 1 are pleiotropic, causing multisystem pathologies rather than isolated abnormalities involving spermatogenesis. For example, the autosomal recessive mutation sks (skeletal fusions with sterility) results in arrest of germ cell development at late meiotic prophase but also causes skeletal fusions of vertebrae and ribs, resulting in body shortening and tail kinks (Vogelweid et al., 1939). In many of these mutations, the effects on germ cells are broad and include both reduced cell numbers and abnormal cell morphologies. The genes associated with infertility have various and sundry functions and include metabolic proteins (ornithine decarboxylase), heat shock proteins/molecular chaperones proteins (Hsp70-2), transcriptional activators (CREM), and proteins involved in DNA repair and maintenance of genome stability, such as the DNA mismatch repair homologue pms2 and the p53 gene (Baker et al., 1995; Donehower et al., 1992).
1.2.3 Spermatogenesis and DNA Repair Mutations
Some of the best characterized genes required for spermatogenesis are those involved in DNA repair. DNA repair defects that delay or prevent the completion of meiotic recombination lead to disruption of the meiotic process (Baker et al., 1995; Arnheim and Shibata, 1997; Edelmann et al., 1996; Hawley and Friend, 1996; Kolodner, 1995; McKee, 1996; Modrich and Lahue, 1996; Rose and Holm, 1993), typically resulting in arrest at the pachytene stage. Factors have been identified which are exclusively required for meiotic events and others which play roles in both mitotic and meiotic cells. Topoisomerase II conditional mutants are one example of the latter. These mutants exhibit both enhanced mitotic recombination and meiotic pachytene arrest (Rose and Holm, 1993). A number of recent reviews have described how eukaryotes maintain chromosome integrity in meiotic cells through DNA repair (Kleckner, 1996; Stahl, 1996; Roeder, 1997). Meiotic cells seem to have developed mechanisms functionally equivalent to mitotic cell cycle checkpoints to sense DNA strand breaks and prevent cells from progressing through the cell cycle until DNA damage is resolved. In yeast it has been shown that several of the proteins involved in mitotic DNA strand-break cell cycle checkpoints (Rad17, Rad 24 and Mec1) are necessary for preventing cells from progressing into meiotic division I before recombination is complete (Handel, 1987). Based on these and similar observations, it has been proposed that the meiotic cell cycle is an evolutionary product of mitosis, with diploidy and strand break-sensing checkpoint mechanisms serving to ensure the integrity of the genome (Kleckner, 1996).
An interesting feature of mice missing various DNA repair components is that many targeted mutations that disrupt normal DNA repair and genome stability genes also have profound effects on germ cell development and spermatogenesis (Arnheim and Shibata, 1997)(Table 2). Some of these mutations involve defects in gametogenesis in both sexes (for example the mlh1 and Atmxe2x88x92/xe2x88x92mice), while deficiencies in other genes cause male germ cell arrest specifically (pms2xe2x88x92/xe2x88x92mice). Clearly male and female gametogenesis are biologically different processes although the molecular basis for the difference is not known.
1.2.4 Drosophila Spermatogenesis Genes
Many insights into germ cell development have come from utilizing Drosophila melanogaster and Drosophila hydei as model systems. Drosophila has a number of advantages over other organisms for studies of germ cells, including the ease of generating large numbers of new mutants tagged with selectable transposable elements, well defined developmental germ cell stages (Lindsley and Tokuyasu, 1980) with characterized mutants at each stage, and a small genome with few chromosomes to simplify genetic and cytogenetic analyses. These advantages have led to the generation of a significant number of mutants that interrupt gametogenesis at various stages (Maines and Wasserman, 1998; Williamson and Lehmann, 1996).
Analysis of several Drosophila spermatogenesis mutants has defined stage specific defects that can be grouped into proliferative, growth phase, meiotic and postmeiotic mutants (Castrillon et al., 1993). Several mutants interrupt spermatogenesis at the mitotic to meiotic transition.
The twine mutant affects a cell cycle cdc2 homologue that is expressed specifically in germ cells (Alphey et al., 1992; Courtot et al., 1992). Twine arrests development at the 16 cell stage in spermatocyte cysts, but interestingly, some spermatid cell differentiation continues in these tetraploid cells. Twine seems to be involved in preparation of chromosomes in both males and females for the two meiotic reduction divisions (Eberhart and Wasserman, 1995; White-Cooper et al., 1993). Two other mutants, pelota and boule, were identified in a screen for viable male sterile mutations. Like twine, pelota arrests the meiotic reduction process but allows some differentiation to proceed, albeit aberrant (Castrillon et al., 1993; Eberhart and Wasserman, 1995). Boule has a similar phenotype to pelota (Eberhart et al., 1996), but is unique in that its expression is testes specific (Shan et al., 1996), and it has a human homologue, DAZ, which is frequently deleted in azoospermic men (Reijo et al., 1995; Simoni et al., 1997).
Four separate spermatogenesis mutants: always early, cannonball, meiosis I arrest and spermatocyte arrest have also been identified. These mutations are unique in that they arrest both spermatogenesis at the mitotic to meiotic transition and also block postmeiotic differentiation, in contrast to twine, pelota and boule (Lin et al., 1996). Mutations at the bocce locus produce cysts that have primary spermatocytes in variable number. However progression to meiotic cysts is generally arrested, with only rare postmeiotic cysts present (Castrillon et al., 1993).
There are several significant differences between Drosophila and mammalian spermatogenesis. These differences include: 1) the absence of meiotic recombination in Drosophila males, 2) the ability of Drosophila germ cells to progress through spermatogenesis despite the cessation of transcription at entry into meiosis (Brink, 1968), 3) the presence of apbptosis during normal germ cell differentiation in rodents and humans, and 4) the protracted duration of mammalian meiosis. Teleologically, these differences may serve to limit the number of defective gametes in mammals, in whom small numbers of progeny may impose strong selection against production of zygotes with DNA mutations or chromosomal abnormalities.
1.3 Deficiencies in the Prior Art
There remains a need in the art for polynucleotide and polypeptide compositions useful in the diagnosis and treatment of male infertility, defects in spermatogenesis, and in the detection and treatment of male cancers, and in particular, testicular cancer and related disorders. The identification and characterization of male germ cell specific nucleic acid and amino acid compositions that are involved in spermatogenesis, particularly in the progression through meiosis, would represent a significant advance in the art.
The present invention overcomes these and other limitations in the prior art by providing a novel male germ cell polypeptide (designated MORC) and the gene which encodes it (designated MORC). The gene encoding the murine MORC polypeptide is given in SEQ ID NO:1, with the corresponding murine MORC polypeptide shown in SEQ ID NO:2. The gene encoding the human MORC polypeptide is given in SEQ ID NO:3, with the corresponding human MORC polypeptide shown in SEQ ID NO:4.
The present invention provides an isolated nucleic acid segment comprising a gene encoding a MORC polypeptide, wherein the MORC polypeptide comprises a contiguous amino acid sequence of at least 27 amino acids SEQ ID NO:2 or SEQ ID NO:4. In certain preferred embodiments, the MORC polypeptide comprises the contiguous amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
In particular aspects, the gene comprises the nucleic acid sequence from position 105 to position 2954 of SEQ ID NO:1, or the nucleic acid sequence from position 63 to position 3014 of SEQ ID NO:3, or a complement thereof, or a sequence that hybridizes to the sequence from position 105 to position 2954 of SEQ ID NO:1, or the nucleic acid sequence from position 63 to position 3014 of SEQ ID NO:3, under conditions of high stringency.
In preferred embodiments of the invention, the nucleic acid segment is isolated from a mammalian cell, including, but not limited to, a rodent cell, such as a mouse or a rat cell, or in particularly preferred aspects, a human cell.
In further aspects of the invention, the nucleic acid segment is operably linked to a promoter that directs the expression of the nucleic acid segment in a host cell. In certain aspects, the promoter is a heterologous promoter.
In other preferred embodiments of the present invention, the isolated nucleic acid segment is comprised within a recombinant vector. Exemplary recombinant vectors include, but are not limited to, artificial chromosome vectors such as bacterial artificial chromosomes and yeast artificial chromosomes, viral vectors such as adenoviral, adeno-associated viral, retroviral, herpes viral, vaccinia viral or baculoviral vectors, plasmids, cosmids and phagemids.
The present invention also provides an isolated nucleic acid segment characterized as an isolated nucleic acid segment comprising a sequence region that consists of at least 23 contiguous nucleotides that have the same sequence as, or are complementary to, at least 23 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3; or an isolated nucleic acid segment of from 23 to about 20,000 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:1 or SEQ ID NO:3; or the complement thereof, under stringent hybridization conditions.
In certain aspects of the invention, the nucleic acid segment comprises a sequence region of at least 30 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 250 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides or at least 2500 nucleotides, or the nucleic acid segment is 30, 50, 100, 250, 500, 1000, 2000 or 2500 nucleotides in length. In particular aspects, the nucleic acid segment comprises the sequence of SEQ ID NO:1 or SEQ ID NO:3. In other aspects of the invention, the nucleic acid is up to 10,000, up to 5,000 or up to 3,000 base pairs in length.
A further objective of the invention is to provide polynucleotide segments comprising all or parts of a gene encoding MORC. Polynucleotide probes and primers specific for these MORC. genes also represent important compositions provided by the invention.
The present invention further provides a recombinant host cell comprising an isolated nucleic acid segment comprising a gene encoding a MORC polypeptide, wherein the MORC polypeptide comprises a contiguous amino acid sequence of at least 27 amino acids SEQ ID NO:2 or SEQ ID NO:4. In preferred aspects, the recombinant host cell is further defined as a prokaryotic cell, such as a bacterial cell or, more particularly, an E. coli cell. In other preferred aspects, the recombinant host cell is further defined as a eukaryotic cell, such as an animal cell or a fungal cell. In particularly preferred embodiments, the animal cell is a mammalian cell, exemplified by, but not limited to, a human, mouse, rat, monkey, chicken, dog, cat, horse, pig, cow, sheep, goat or hamster cell. In still other preferred aspects, the mammalian cell is a tumor cell, such as a testicular cancer cell.
In further embodiments, the isolated nucleic acid segment is introduced into the cell by a recombinant vector, wherein the host cell expresses the isolated nucleic acid segment to produce a MORC peptide or polypeptide. In additional aspects, the MORC peptide or polypeptide comprises a contiguous amino acid sequence of at least about 27 amino acids from SEQ ID NO:2 or SEQ ID NO:4.
Another aspect of the present invention is an animal cell, such as a human or other animal cell, that comprises a MORC polypeptide or polynucleotide. In a preferred embodiment, the cell is a mouse male germ cell that produces a MORC polypeptide of approximately 108-kDa, and that is identical to, or substantially homologous with, the MORC polypeptide identified in SEQ ID NO:2, or a human male germ cell that produces a MORC polypeptide of approximately 118-kDa, and that is identical to, or substantially homologous with, the MORC polypeptide identified in SEQ ID NO:4.
A further aspect of the present invention is a vector (such as a plasmid, cosmid, virus, phagemid, or the like), that includes within its nucleotide sequence a nucleic acid segment that comprises one or more MORC genes, or portions thereof. Preferably such a vector is comprised within a transformed host cell. The transformed host cell may be a bacterial, animal, fungal, or plant cell, and may be comprised within a transgenic animal, or may be comprised within a culture of bacteria, yeast, fungus, animal or plant cells.
Another embodiment of the invention provides polypeptides and peptides comprising at least 26 or more, and preferably, substantially all of the amino acid sequences disclosed in SEQ ID NO:2 and SEQ ID NO:4. Polypeptides having MORC activity represent important compositions provided by the invention. It is a further objective of the invention to provide methods for identifying MORC polypeptide and polynucleotide compositions, methods for producing such compositions, and methods for using these compositions in a variety of diagnostic and therapeutic regimens. The invention also provides methods and compositions for the detection of MORC compositions in biological and clinical samples.
The present invention also provides a method of preparing a MORC peptide or polypeptide, comprising the steps of expressing an isolated nucleic acid segment comprising a MORC gene encoding a MORC polypeptide, wherein the MORC polypeptide comprises a contiguous amino acid sequence of at least 27 amino acids SEQ ID NO:2 or SEQ ID NO:4, the nucleic acid segment operably linked to a promoter, in a host cell, and collecting the MORC peptide or polypeptide so expressed.
Also provided are nucleic acid detection kits comprising, in a suitable container, an isolated nucleic acid segment characterized as an isolated nucleic acid segment comprising a sequence region that consists of at least 23 contiguous nucleotides that have the same sequence as, or are complementary to, at least 23 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3; or an isolated nucleic acid segment of from 23 to about 20,000 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:1 or SEQ ID NO:3; or the complement thereof, under stringent hybridization conditions, and a detection reagent.
Thus, the present invention provides a method for detecting a nucleic acid sequence encoding a MORC polypeptide, comprising the steps of contacting sample nucleic acids suspected of encoding a MORC polypeptide with at least a first isolated nucleic acid segment comprising a nucleic acid sequence of at least about 23 contiguous nucleotides of from position 105 to position 2954 of SEQ ID NO:1 or from position 63 to position 3014 of SEQ ID NO:3 under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting the hybridized complementary nucleic acids thus formed, wherein the presence of hybridized complementary nucleic acids is indicative of the presence of a nucleic acid sequence encoding a MORC polypeptide in the sample nucleic acids. In certain aspects, the sample nucleic acids contacted are located within a cell, while in other aspects, the sample nucleic acids are separated from a cell prior to contact. In further aspects, the isolated nucleic acid comprises a detectable label and the complex is detected by detecting the label.
In another embodiment, there is provided a monoclonal antibody that binds immunologically to a male germ cell polypeptide designated as MORC. The antibody may be non-cross reactive with other human polypeptides, or it may bind to non-human MORC, but not to human MORC. The antibody may further comprise a detectable label, such as a fluorescent label, a chemiluminescent label, a radiolabel or an enzyme. Also encompassed are hybridoma cells and cell lines producing such antibodies.
In another embodiment, there is included a polyclonal antisera, antibodies of which bind immunologically to a MORC polypeptide. The antisera may be derived from any animal, but preferably is from an animal other than a human. Preferred antigens for the preparation of such sera include a MORC polypeptide isolated from a human, rat, goat, rabbit, pig, horse, cat, dog, hamster, monkey or other such animal cell line. Preferred hosts for the preparation of a polyclonal antisera specific for MORC include animals such as rabbits, goats, and other such animals.
The invention also provides pharmaceutical compositions which comprise one or more of the MORC compositions disclosed herein. Such compositions may include MORC or MORC-derived polypeptides, polynucleotides, antisense oligonucleotides, ribozymes, antibodies, antisera, antigens, peptide epitopes, protein fusions, peptides and the like.
The present invention also provides a purified polypeptide comprising a sequence region of at least 27, at least 30, at least 35, at least 50, at least 100, at least 200, at least 500 or at least 900 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:4. In certain aspects of the invention, the purified polypeptide comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4. In these aspects of the invention, the polypeptide may be encoded by the nucleic acid sequence from position 105 to position 2954 of SEQ ID NO:1, or the nucleic acid sequence from position 63 to position 3014 of SEQ ID NO:3. In certain preferred aspects of the invention, the purified polypeptide is operatively linked to a selected amino acid sequence.
Thus, the present invention further provides a fusion protein comprising a purified polypeptide comprising a sequence region of at least 27 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:4, operatively linked to a selected amino acid sequence. In particular embodiments, the selected amino acid sequence is an antigenic amino acid sequence.
The present invention also provides an antibody that specifically binds to a polypeptide comprising a sequence region of at least 27 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:4. In preferred embodiments, the antibody is a monoclonal antibody.
The present invention further provides a method for detecting a MORC polypeptide in a biological sample, comprising the steps of contacting a biological sample suspected of containing a MORC polypeptide with at least a first antibody that specifically binds to a MORC peptide or polypeptide, under conditions effective to allow the formation of complexes, and detecting the complexes so formed.
Additionally provided are immunodetection kits comprising, in a suitable container, an antibody that specifically binds to a polypeptide comprising a sequence region of at least 27 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:4, and an immunodetection reagent.
In still yet a further embodiment, there is provided transgenic mammal in which both copies of the native MORC gene are interrupted or replaced with another gene.
The present invention also provides a transgenic non-human animal having incorporated into its genome a polynucleotide comprising a transgene that encodes a polypeptide comprising a sequence region of at least 27 contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:4.
Also provided is a composition comprising a MORC polypeptide prepared by a process comprising the steps of culturing a host cell comprising a nucleic acid segment encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 under conditions effective to produce the polypeptide, and obtaining the MORC polypeptide from the cell.
The invention also provides a method of identifying a male patient having or at risk for developing infertility, comprising determining the amount of a MORC composition present within a biological sample from the male patient, wherein the absence of the MORC composition in comparison to a sample from a normal male subject, is indicative of a male patient having or at risk for developing infertility. The invention also provides a method of treating infertility, comprising administering to a patient in need of infertility treatment a therapeutically effective amount of a MORC composition.
The invention further provides a method of identifying a male patient having or at risk for developing a germ cell cancer, such as testicular cancer, comprising determining the amount of a MORC composition present within a biological sample from the male patient, wherein the absence of the MORC composition in comparison to a sample from a normal male subject, is indicative of a male patient having or at risk for developing a germ cell cancer, such as testicular cancer.
The invention also provides a method of treating a germ cell cancer, such as testicular cancer, comprising administering to a patient in need of treatment a therapeutically effective amount of a MORC composition. The invention further provides a method of treating a subject having cancer comprising administering contacting a cancer cell within the subject with an expression vector comprising a nucleic acid segment encoding an MORC polypeptide under the transcriptional control of a promoter, wherein expression of the MORC polypeptide is at a level effective to confer a therapeutic benefit on the subject. Treatment of any subject with cancer, including humans, is contemplated. Moreover, in another aspect of the invention, methods of treating cancer also include contacting a cancer cell with an anticancer agent. Anticancer agents include those agents used in chemotherapy, radiotherapy, immunotherapy, and gene therapy for the treatment of cancer. A combination gene therapy approach is also provided by the present invention. For example, a MORC polypeptide and a 123F2 polypeptide, which interacts with a MORC polypeptide, may be provided simultaneously or sequentially to a cancer cell as a combination therapy.
In other embodiments of the present invention, screening methods to identify compounds that are involved in spermatogenesis or cancer are provided. The MORC polypeptide interacts with a 123F2 polypeptide, and the 123F2 polypeptide relocalizes MORC from the nucleus to the cytoplasm. Such methods concern finding compounds that modulate the interaction between a MORC polypeptide and a 123F2 polypeptide. These compounds would then be implicated in either the cancer process or spermatogenesis.
The present invention also provides a method of treating microrchidia, comprising administering to a patient in need of treatment a therapeutically effective amount of a MORC composition. Further, the present invention provides a method of altering spermatogenesis, comprising administering to a patient in need of spermatogenesis alteration a therapeutically effective amount of a MORC composition. Also, the invention provides a method of male contraception, comprising administering to a male animal in need of contraception a biologically effective amount of a MORC composition.
The foregoing objects of the invention and others that are now readily apparent to those of skill in the art having the benefit of the present disclosure are described more fully in the sections which follow:
In one embodiment, the present invention concerns DNA segments, that can be isolated from virtually any source, that are free from total genomic DNA and that encode the whole or a portion of the novel peptide disclosed herein. The murine MORC gene (position 105 to position 2954 of SEQ ID NO:1) encodes a MORC polypeptide having the contiguous amino acid sequence shown in SEQ ID NO:2. The human MORC gene (position 63 to position 3014 of SEQ ID NO:3) encodes a MORC polypeptide having the contiguous amino acid sequence shown in SEQ ID NO:4. The inventors contemplate a variety of MORC DNA segments from the present invention will find particular utility. For example, those segments that encode all or portions of the MORC polypeptide, or subunits, functional domains, and the like of MORC and MORC-related polypeptides, or those segments that comprise one or more MORC promoter or enhancer regions will be useful in a variety of diagnostic, and therapeutic regimens. Such DNA segments may be native DNA segments isolated using molecular biological methods, or alternatively, such segments may be mutagenized segments, or even segments which have been synthesized in vitro either partially or entirely, using chemical synthesis methods that are well-known to those of skill in the art.
As used herein, the term xe2x80x9cDNA segment xe2x80x9d refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a MORC polypeptide or peptide refers to a DNA segment that contains a MORC polypeptide-coding sequence yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term xe2x80x9cDNA segment xe2x80x9d, are DNA segments comprising one or more entire MORC genes and/or promoter regions, as well as all partial and smaller fragments and subfragments isolatable from such entire gene-comprising segments, and also recombinant vectors (such as plasmids, cosmids, phagemids, phage, viruses, and the like) which comprise one or more of the MORC-specific polynucleotide sequences of the invention.
Similarly, a DNA segment comprising an isolated or purified MORC polypeptide-encoding gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term xe2x80x9cgene xe2x80x9d is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional term includes not only genomic sequences, including extrachromosomal DNA sequences, but also operon sequences and/or engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides.
xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case, a MORC gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a MORC polypeptide that includes within its amino acid sequence an at least ten amino acid contiguous sequence from SEQ ID NO:2 or SEQ ID NO:4, and more preferably still, a polypeptide that includes within its amino acid sequence a sequence essentially as set forth in SEQ ID NO:2 or SEQ ID NO:4. In a preferred embodiment, such a DNA segment comprises a gene encoding the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, and more preferably still, comprises a polynucleotide which is identical to, or substantially homologous with, a contiguous polynucleotide sequence from SEQ ID NO:1 or SEQ ID NO:3.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:2 or SEQ ID NO:4,xe2x80x9d means that the sequence substantially corresponds to a portion of the sequence of SEQ ID NO:2 or SEQ ID NO:4 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% amino acid sequence identity or functional equivalence to the amino acids of SEQ ID NO:2 or SEQ ID NO:4 will be sequences that are xe2x80x9cessentially as set forth in SEQ ID NO:2 or SEQ ID NO:4.xe2x80x9d
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch encoding the whole or a portion of the peptide sequence disclosed in SEQ ID NO:2 or SEQ ID NO:4, or that are identical to or complementary to DNA sequences which encode the peptide disclosed in SEQ ID NO:2 or SEQ ID NO:4, and particularly the DNA segment disclosed in either of SEQ ID NO:1 or SEQ ID NO:3. For example, DNA sequences such as about 23 nucleotides, and that are up to about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50, and about 23 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
It will be readily understood that xe2x80x9cintermediate lengthsxe2x80x9d, in these contexts, means any. length between the quoted ranges, such as 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up to and including sequences of about 6,000, 7,000, 8,000, 9,000, 10,000, 10,001, 10002, 10003, 11000, 12000, 13000, or so nucleotides and the like.
It will also be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, including the DNA sequence which is particularly disclosed in SEQ ID NO:1 and SEQ ID NO:3. Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
The DNA segments of the present invention encompass biologically-functional, equivalent peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally-equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein.
In addition to their use in directing the expression of the gene product of the novel MORC genes of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 23 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 23 nucleotide long contiguous DNA segment of SEQ ID NO:1 or SEQ ID NO:3 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10000 etc. (including all intermediate lengths and up to and including full-length sequences) will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to MORC and MORC-related gene sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or even of 100-200 nucleotides or so, identical or complementary to the DNA sequence of SEQ ID NO:1 or SEQ ID NO:3, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 23 and about 100 or so nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 23 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 23 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 23 to 35 contiguous nucleotides, or even longer where desired.
Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR(trademark) technology of U.S. Pat. Nos. 4,683,195 and 4,683,202 (each incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50xc2x0 C. to about 70xc2x0 C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating polynucleotide segments comprising MORC or MORC-related gene(s). Detection of DNA segments via hybridization is well-known to those of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al., 1994; Segal 1976; Prokop, 1991; and Kuby, 1991, are particularly relevant.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate MORC polypeptide-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20xc2x0 C. to about 55xc2x0 C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.
The invention also discloses and claims a composition comprising a MORC or MORC-related polypeptide. The composition may comprises one or more host cells which express a MORC or MORC-related polypeptide, recombinant host cells expresses the protein, cell suspensions, extracts, inclusion bodies, or tissue cultures or culture extracts which contain the MORC protein, culture supernatant, disrupted cells, cell extracts, lysates, homogenates, and the like. The compositions may be in aqueous form, or alternatively, in dry, semi-wet, or similar forms such as cell paste, cell pellets, or alternatively freeze dried, powdered, lyophilized, evaporated, or otherwise similarly prepared in dry form. Such means for preparing MORC polypeptides are well-known to those of skill in the art of protein isolation and purification. In certain embodiments, the MORC polypeptides may be purified, concentrated, admixed with other reagents, or processed to a desired final form. Preferably, the composition will comprise from about 1% to about 90% by weight of the MORC polypeptide, and more preferably from about 5% to about 50% by weight.
In a preferred embodiment, the MORC polypeptide compositions of the invention may be prepared by a process which comprises the steps of culturing a host cell which expresses a MORC or MORC-related polypeptide under conditions effective to produce such a protein, and then obtaining the protein from the cell. The obtaining of such a MORC polypeptide may further include purifying, concentrating, processing, or admixing the protein with one or more reagents. Preferably, the MORC or MORC-related polypeptide is obtained in an amount of from between about 1% to about 90% by weight, and more preferably from about 5% to about 70% by weight, and even more preferably from about 10% to about 20% to about 30%, or even to about 40% or 50% by weight.
The invention also relates to a method of preparing a MORC polypeptide composition. Such a method generally involves the steps of culturing a host cell which expresses a MORC polypeptide under conditions effective to produce the protein, and then obtaining the protein so produced. In a preferred embodiment the cell is an male germ cell, or any recombinant host cell which contains a MORC-encoding DNA segment. Alternatively, the recombinant plasmid vectors of the invention may be used to transform other suitable bacterial or eukaryotic cells to produce the MORC polypeptide of the invention. Eukaryotic host cells including human, mouse, and monkey, as well as yeast cells are contemplated to be particularly useful in the preparation of the MORC protein. Likewise, prokaryotic host cells including Gram-negative cells such as E. coli, Pseudomonas spp. and related Enterobacteraceae and the like are all contemplated to be useful in the preparation of the MORC polypeptides of the invention.
In such embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a MORC polypeptide or peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, or eukaryotic cell. Preferred eukaryotic cells are animal cells, with mammalian cells, particularly human cells, being most preferred. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, tissue, organism, animal, or recombinant host cell chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of polypeptides or epitopic core regions, such as may be used to generate anti-MORC antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful. Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequences from SEQ ID NO:2 or SEQ ID NO:4.
In yet another aspect, the present invention provides methods for producing a transgenic cell, and in particular a plant or animal cell which expresses a nucleic acid segment encoding the novel MORC polypeptide of the present invention. The process of producing transgenic cells is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operatively linked to a coding region that encodes a MORC polypeptide. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant MORC polypeptide expressed in a particular transgenic cell, the invention also provides for the expression of MORC-specific mRNA, and antisense polynucleotides that specifically bind to MORC-specific mRNA. The use of antisense compositions as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art, and is described in detail herein.
In a preferred embodiment, the invention encompasses an animal cell which has been transformed with a nucleic acid segment of the invention, and which expresses a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein. As used herein, the term xe2x80x9ctransgenic host cellxe2x80x9d is intended to refer to a host cell, either prokaryotic or eukaryotic, that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein (xe2x80x9cexpressedxe2x80x9d), or any other genes or DNA sequences which one desires to introduce into the non-transformed host cell, such as genes which may normally be present in the non-transformed cell but which one desires to either genetically engineer or to have altered expression.
It is contemplated that in some instances the genome of a transgenic host cell of the present invention will have been augmented through the stable introduction of a MORC transgene, either-native MORC, or synthetically modified or mutated MORC. In some instances, more than one transgene will be incorporated into the genome of the transformed host cell. Such is the case when more than one MORC polypeptide-encoding DNA segment is incorporated into the genome of such a cell. In certain situations, it may be desirable to have one, two, three, four, or even more MORC polypeptides (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic host cell. In preferred embodiments, the introduction of the transgene into the genome of the host cell results in a stable integration wherein the progeny of such cells also contain a copy of the transgene in their genome.
A preferred gene which may be introduced includes, for example, a DNA segment comprising one or more MORC gene(s), and particularly one or more of the MORC or MORC-like polypeptides disclosed herein. Highly preferred nucleic acid sequences are those which have the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3, or biologically-functional equivalents thereof, sequences which hybridize to the sequence of SEQ ID NO:1 or SEQ ID NO:3, or sequences which encode the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or sequences which encode a biologically functional equivalent protein of SEQ ID NO:2 or SEQ ID NO:4, or any of those sequences which have been genetically engineered to alter, modify, change, decrease or increase the suppressor activity or specificity of the MORC polypeptide in such a transformed host cell.
Means for transforming a host cell and the preparation of a transgenic cell line are well-known in the art (as exemplified in U.S. Pat. Nos. 5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and 5,225,341, all specifically incorporated herein by reference), and are briefly discussed herein. Vectors, including plasmids, cosmids, phage, phagemids, BACs (bacterial artificial chromosomes), YACs (yeast artificial chromosomes), and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed polypeptides. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively- or negatively-regulating activity upon the particular genes of interest as desired. The DNA segment or gene may encode either a native or modified polypeptide, which will be expressed in the resultant recombinant cells, and/or which will impart a desired phenotype to the transformed host cell.
In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which specifically bind to one or more of the MORC polypeptides disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g, Harlow and Lane, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund""s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund""s adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified MORC polypeptide, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5xc3x97107 to 2xc3x97108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
Fusion procedures usually produce viable hybrids at low frequencies, about 1xc3x9710xe2x88x926 to 1xc3x9710xe2x88x928. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two wk. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three wk) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
The present invention also provides compositions, methods and kits for screening samples suspected of containing a MORC polypeptide or a MORC polynucleotide that encodes such a polypeptide. Alternatively, the invention provides compositions, methods and kits for screening samples suspected of containing MORC or MORC-related polypeptides or genes encoding MORC or MORC-related polypeptides which are functionally equivalent to, or substantially homologous to, the MORC polypeptides disclosed herein. Such screening may be performed on samples such as transformed host cells, clinical or laboratory samples suspected of containing or producing such a polypeptide or nucleic acid segment. A kit can contain a novel nucleic acid segment or an antibody of the present invention. The kit can contain reagents for detecting an interaction between a sample and a nucleic acid or an antibody of the present invention. The provided reagent can be radio-, fluorescently- or enzymatically-labeled. The kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.
The reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is proposed that the MORC polypeptides or peptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect MORC or MORC-related polypeptides, peptides, or epitope-containing sequences specific to MORC. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.
In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like. Generally, immunocomplex formation will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
For assaying purposes, it is proposed that virtually any sample suspected of comprising either a MORC polypeptide, a MORC-derived peptide or a MORC-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of MORC polypeptides or related peptides and/or antibodies in a sample. Samples may include cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing MORC polypeptides or peptide fragments thereof. Generally speaking, kits in accordance with the present invention will include a suitable MORC polypeptide, peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
The present invention is also directed to MORC protein or peptide compositions, free from total cells and other peptides, which comprise a purified MORC protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more anti-MORC antibodies. In particular, the invention concerns epitopic core sequences derived from MORC and MORC-derived proteins or peptides.
As used herein, the term xe2x80x9cincorporating an epitope(s) that is immunologically cross-reactive with one or more anti-MORC antibodiesxe2x80x9d is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a MORC polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the MORC polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
The identification of MORC immunodominant epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, e.g., Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these xe2x80x9cepitopic core sequencesxe2x80x9d may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
Preferred peptides for use in accordance with the present invention will generally be on the order of about 8 to about 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic MORC-derived peptides will provide advantages in certain circumstances, for example, in the preparation of immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a xe2x80x9cuniversalxe2x80x9d epitopic peptide directed to MORC polypeptides, and in particular mammalian MORC and/or MORC-related polypeptide sequences. These epitopic core sequences are identified herein in particular aspects as hydrophilic regions of the particular polypeptide antigen. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation, and, hence, elicit specific antibody production.
An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is xe2x80x9ccomplementaryxe2x80x9d to, and therefore will bind, antigen binding sites on the MORC polypeptide-directed antibodies disclosed herein. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term xe2x80x9ccomplementaryxe2x80x9d refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 8 amino acids in length, with sequences on the order of 10 to 20 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysis programs (e.g., DNAStar(copyright) software, DNAStar, Inc., Madison, Wis.) may also be useful in designing synthetic peptides in accordance with the present disclosure.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at about 4xc2x0 C., or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.